The discovery of small non-coding RNAs (sncRNAs) with regulatory functions is a recent breakthrough in biology. Among sncRNAs, microRNAs (miRNAs) and virus-derived sncRNAs have emerged as elements of critical importance in controlling viral replication and host responses to viral infection. However, the expression patterns and functional aspects of other types of sncRNAs after viral infection are completely unexplored. To define the expression patterns of sncRNAs, as well as to discover novel regulatory sncRNAs in response to viral infection, we applied next-generation sequencing (NGS) to cells infected with human respiratory syncytial virus (RSV), the most common respiratory mucosal virus that causes lung and airway infections in infants and young children, in comparison to uninfected cells. We found that RSV infection leads to abundant production of tRNA-derived RNA fragments (tRFs) that are approximately 30 nucleotides (nts) long and correspond to the 5'- half of mature tRNAs (tRF5). The induction of tRF5 is virus-specific and RSV replication-dependent. At least one member of these tRF5s, which is derived from tRNA-Glu-CTC (tRF5-GluCTC), decreases the level of target mRNAs in the cytoplasm. It also plays a critical role in RSV infection, as RSV replication is attenuated by its suppression, but promoted by its ectopic expression. The biogenesis of this tRF is also specific, and mediated by a particular endonuclease (angiogenin, ANG), and not by other nucleases. The central hypothesis of this project is that tRFs are not a random by-product of tRNA degradation, but rather are functional molecules. This central hypothesis will be addressed by identifying the targets of tRF5-GluCTC in RSV-infected airway epithelial cells (Aim 1), determining the viral factor(s) controlling the trans-silencing activity of tRFs (Aim 2), and defining the viral component(s) contributing to ANG-mediated biogenesis of tRFs (Aim 3). Preliminary data suggest that the RSV proteins N and P will be a focus of Aims 2 and 3, respectively. Of note, despite the public health importance of RSV, no effective therapeutic interventions or vaccines are available. Therefore, the overall goal of this project is to use a combination of molecular virology, protein and RNA biochemistry, cellular and structural biology techniques, to identify the molecular mechanisms underlying the regulatory effects of this novel tRF on RSV replication. The results of this project will provide an important new perspective and novel regulatory mechanisms to study the interactions between the host and RSV, and lay the foundation for the future development of biomarkers, therapeutic interventions, and vaccine designs based on the interaction of tRFs with their targets, both host and virus-derived, and the biogenic mechanisms of tRFs.